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Microglial Keratan Sulfate Epitope Elicits in Central Nervous Tissues of Transgenic Model Mice and Patients with Amyotrophic Lateral Sclerosis

2015; Elsevier BV; Volume: 185; Issue: 11 Linguagem: Inglês

10.1016/j.ajpath.2015.07.016

ISSN

1525-2191

Autores

Tahmina Foyez, Yoshiko Takeda‐Uchimura, Shinsuke Ishigaki, Narentuya, Zui Zhang, Gen Sobue, Kenji Kadomatsu, Kenji Uchimura,

Tópico(s)

Nerve injury and regeneration

Resumo

The functional role of 5D4 antibody-reactive keratan sulfate (KS) in the pathogenesis of neurodegenerative diseases is unknown. We therefore studied the expression of 5D4-reactive KS in amyotrophic lateral sclerosis (ALS), a motor neuron-degenerative disease, with the use of SOD1G93A ALS model mice and patients with ALS. Histochemical and immunoelectron microscopic characterizations showed that the 5D4-reactive KS is expressed in Mac2/galectin-3–positive activated or proliferating microglia of SOD1G93A ALS model mice at disease end stage and that the KS is an O-linked glycan modified with sialic acid and fucose, which was thus far shown to exist in cartilage. Intriguingly, microglial KS was detected in the spinal cord and brainstem but not in the cerebral cortex of SOD1G93A mice. We found that KSGal6ST, a galactose-6-sulfotransferase, is required for biosynthesis of the microglial 5D4-reactive KS by generating SOD1G93A/KSGal6ST−/− mice. The requirement of GlcNAc6ST1 for this synthesis was corroborated by analyzing SOD1G93A/GlcNAc6ST1−/− mice. These results indicate that both galactose-6– and N acteylglucosamine-6–sulfated KS elicited in the spinal cord and brainstem are associated with the degeneration of spinal and bulbar lower motor neurons in ALS pathology and may play a role in disease progression via microglial activation and proliferation. The functional role of 5D4 antibody-reactive keratan sulfate (KS) in the pathogenesis of neurodegenerative diseases is unknown. We therefore studied the expression of 5D4-reactive KS in amyotrophic lateral sclerosis (ALS), a motor neuron-degenerative disease, with the use of SOD1G93A ALS model mice and patients with ALS. Histochemical and immunoelectron microscopic characterizations showed that the 5D4-reactive KS is expressed in Mac2/galectin-3–positive activated or proliferating microglia of SOD1G93A ALS model mice at disease end stage and that the KS is an O-linked glycan modified with sialic acid and fucose, which was thus far shown to exist in cartilage. Intriguingly, microglial KS was detected in the spinal cord and brainstem but not in the cerebral cortex of SOD1G93A mice. We found that KSGal6ST, a galactose-6-sulfotransferase, is required for biosynthesis of the microglial 5D4-reactive KS by generating SOD1G93A/KSGal6ST−/− mice. The requirement of GlcNAc6ST1 for this synthesis was corroborated by analyzing SOD1G93A/GlcNAc6ST1−/− mice. These results indicate that both galactose-6– and N acteylglucosamine-6–sulfated KS elicited in the spinal cord and brainstem are associated with the degeneration of spinal and bulbar lower motor neurons in ALS pathology and may play a role in disease progression via microglial activation and proliferation. Glycan structures often show characteristic differences between a normal state and disease. Knowledge of disease-specific glycan structures can help to understand the mechanisms underlying disease progression and to develop new diagnostic and therapeutic approaches. Keratan sulfate (KS) is an extracellular polysaccharide classified as a member of the glycosaminoglycans. It was originally isolated from the cornea.1Meyer K. Linker A. Davidson E.A. Weissmann B. The mucopolysaccharides of bovine cornea.J Biol Chem. 1953; 205: 611-616Abstract Full Text PDF PubMed Google Scholar KS is also found in skeletal and nervous tissues.2Funderburgh J.L. Keratan sulfate: structure, biosynthesis, and function.Glycobiology. 2000; 10: 951-958Crossref PubMed Scopus (308) Google Scholar, 3Kleene R. Schachner M. Glycans and neural cell interactions.Nat Rev Neurosci. 2004; 5: 195-208Crossref PubMed Scopus (406) Google Scholar In terms of the composition of KS proteoglycans, one or more KS chains are covalently attached to a core protein through the elongation of N-linked, O-N-acetylgalactosamine (GalNAc)-linked or O-mannose–linked oligosaccharides.4Funderburgh J.L. Keratan sulfate biosynthesis.IUBMB Life. 2002; 54: 187-194Crossref PubMed Scopus (101) Google Scholar, 5Krusius T. Finne J. Margolis R.K. Margolis R.U. Identification of an O-glycosidic mannose-linked sialylated tetrasaccharide and keratan sulfate oligosaccharides in the chondroitin sulfate proteoglycan of brain.J Biol Chem. 1986; 261: 8237-8242Abstract Full Text PDF PubMed Google Scholar KS is composed of repeating disaccharides of galactose (Gal) and N-acetylglucosamine (GlcNAc), with sulfate groups at their C-6 positions.2Funderburgh J.L. Keratan sulfate: structure, biosynthesis, and function.Glycobiology. 2000; 10: 951-958Crossref PubMed Scopus (308) Google Scholar, 6Uchimura K. Keratan sulfate: biosynthesis, structures, and biological functions.Methods Mol Biol. 2015; 1229: 389-400Crossref PubMed Scopus (22) Google Scholar The capping structures at their nonreducing termini vary and include modifications with α3- or α6-linked sialic acid, α3-linked Gal, or β3-linked sulfated GalNAc.7Tai G.H. Huckerby T.N. Nieduszynski I.A. Multiple non-reducing chain termini isolated from bovine corneal keratan sulfates.J Biol Chem. 1996; 271: 23535-23546Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 8Tai G.H. Nieduszynski I.A. Fullwood N.J. Huckerby T.N. Human corneal keratan sulfates.J Biol Chem. 1997; 272: 28227-28231Crossref PubMed Scopus (37) Google Scholar Golgi-resident enzymes catalyze sulfation modifications and elongation of the KS disaccharides, producing diversity in the chains' structure. The C-6 sulfation modifications in KS synthesis are dictated by the Gal/GlcNAc/GalNAc sulfotransferases.9Uchimura K. Rosen S.D. Sulfated L-selectin ligands as a therapeutic target in chronic inflammation.Trends Immunol. 2006; 27: 559-565Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar Among those, GlcNAc6ST1, GlcNAc6ST3, GlcNAc6ST5, and KS galactose-6-sulfotransferase (KSGal6ST) (encoded by the Chst2, Chst5, CHST6, and Chst1 genes, respectively) were reported to mediate KS sulfation in vivo.10Akama T.O. Nishida K. Nakayama J. Watanabe H. Ozaki K. Nakamura T. Dota A. Kawasaki S. Inoue Y. Maeda N. Yamamoto S. Fujiwara T. Thonar E.J. Shimomura Y. Kinoshita S. Tanigami A. Fukuda M.N. Macular corneal dystrophy type I and type II are caused by distinct mutations in a new sulphotransferase gene.Nat Genet. 2000; 26: 237-241Crossref PubMed Scopus (214) Google Scholar, 11Hayashida Y. Akama T.O. Beecher N. Lewis P. Young R.D. Meek K.M. Kerr B. Hughes C.E. Caterson B. Tanigami A. Nakayama J. Fukada M.N. Tano Y. Nishida K. Quantock A.J. Matrix morphogenesis in cornea is mediated by the modification of keratan sulfate by GlcNAc 6-O-sulfotransferase.Proc Natl Acad Sci U S A. 2006; 103: 13333-13338Crossref PubMed Scopus (57) Google Scholar, 12Hoshino H. Foyez T. Ohtake-Niimi S. Takeda-Uchimura Y. Michikawa M. Kadomatsu K. Uchimura K. KSGal6ST is essential for the 6-sulfation of galactose within keratan sulfate in early postnatal brain.J Histochem Cytochem. 2014; 62: 145-156Crossref PubMed Scopus (22) Google Scholar Several monoclonal antibodies against KS were used to evaluate KS expression.13Caterson B. Christner J.E. Baker J.R. Identification of a monoclonal antibody that specifically recognizes corneal and skeletal keratan sulfate. Monoclonal antibodies to cartilage proteoglycan.J Biol Chem. 1983; 258: 8848-8854Abstract Full Text PDF PubMed Google Scholar, 14Andrews P.W. Banting G. Damjanov I. Arnaud D. Avner P. Three monoclonal antibodies defining distinct differentiation antigens associated with different high molecular weight polypeptides on the surface of human embryonal carcinoma cells.Hybridoma. 1984; 3: 347-361Crossref PubMed Scopus (190) Google Scholar, 15Magro G. Perissinotto D. Schiappacassi M. Goletz S. Otto A. Muller E.C. Bisceglia M. Brown G. Ellis T. Grasso S. Colombatti A. Perris R. Proteomic and postproteomic characterization of keratan sulfate-glycanated isoforms of thyroglobulin and transferrin uniquely elaborated by papillary thyroid carcinomas.Am J Pathol. 2003; 163: 183-196Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 16Kawabe K. Tateyama D. Toyoda H. Kawasaki N. Hashii N. Nakao H. Matsumoto S. Nonaka M. Matsumura H. Hirose Y. Morita A. Katayama M. Sakuma M. Kawasaki N. Furue M.K. Kawasaki T. A novel antibody for human induced pluripotent stem cells and embryonic stem cells recognizes a type of keratan sulfate lacking oversulfated structures.Glycobiology. 2013; 23: 322-336Crossref PubMed Scopus (44) Google Scholar 5D4 recognizes KS oligosaccharide structures with absolute dependence on both Gal- and GlcNAc-6-sulfation modifications.11Hayashida Y. Akama T.O. Beecher N. Lewis P. Young R.D. Meek K.M. Kerr B. Hughes C.E. Caterson B. Tanigami A. Nakayama J. Fukada M.N. Tano Y. Nishida K. Quantock A.J. Matrix morphogenesis in cornea is mediated by the modification of keratan sulfate by GlcNAc 6-O-sulfotransferase.Proc Natl Acad Sci U S A. 2006; 103: 13333-13338Crossref PubMed Scopus (57) Google Scholar, 12Hoshino H. Foyez T. Ohtake-Niimi S. Takeda-Uchimura Y. Michikawa M. Kadomatsu K. Uchimura K. KSGal6ST is essential for the 6-sulfation of galactose within keratan sulfate in early postnatal brain.J Histochem Cytochem. 2014; 62: 145-156Crossref PubMed Scopus (22) Google Scholar, 17Mehmet H. Scudder P. Tang P.W. Hounsell E.F. Caterson B. Feizi T. The antigenic determinants recognized by three monoclonal antibodies to keratan sulphate involve sulphated hepta- or larger oligosaccharides of the poly(N-acetyllactosamine) series.Eur J Biochem. 1986; 157: 385-391Crossref PubMed Scopus (186) Google Scholar, 18Zhang H. Muramatsu T. Murase A. Yuasa S. Uchimura K. Kadomatsu K. N-Acetylglucosamine 6-O-sulfotransferase-1 is required for brain keratan sulfate biosynthesis and glial scar formation after brain injury.Glycobiology. 2006; 16: 702-710Crossref PubMed Scopus (54) Google Scholar However, additional glycosylation modifications of the 5D4-reactive KS glycan and the precise structure of the 5D4 epitope still remain to be determined. 5D4 was raised initially against human articular cartilage proteoglycans.13Caterson B. Christner J.E. Baker J.R. Identification of a monoclonal antibody that specifically recognizes corneal and skeletal keratan sulfate. Monoclonal antibodies to cartilage proteoglycan.J Biol Chem. 1983; 258: 8848-8854Abstract Full Text PDF PubMed Google Scholar In central nervous system tissues, the expression of the 5D4-reactive KS is induced in rodent models after injury.18Zhang H. Muramatsu T. Murase A. Yuasa S. Uchimura K. Kadomatsu K. N-Acetylglucosamine 6-O-sulfotransferase-1 is required for brain keratan sulfate biosynthesis and glial scar formation after brain injury.Glycobiology. 2006; 16: 702-710Crossref PubMed Scopus (54) Google Scholar, 19Jones L.L. Tuszynski M.H. Spinal cord injury elicits expression of keratan sulfate proteoglycans by macrophages, reactive microglia, and oligodendrocyte progenitors.J Neurosci. 2002; 22: 4611-4624Crossref PubMed Google Scholar We have shown that treatment with keratanase II, a KS-degrading bacterial enzyme, or deficiency in the Chst2 gene increased axonal sprouting and regeneration in injured nervous tissues.20Imagama S. Sakamoto K. Tauchi R. Shinjo R. Ohgomori T. Ito Z. Zhang H. Nishida Y. Asami N. Takeshita S. Sugiura N. Watanabe H. Yamashita T. Ishiguro N. Matsuyama Y. Kadomatsu K. Keratan sulfate restricts neural plasticity after spinal cord injury.J Neurosci. 2011; 31: 17091-17102Crossref PubMed Scopus (68) Google Scholar, 21Ito Z. Sakamoto K. Imagama S. Matsuyama Y. Zhang H. Hirano K. Ando K. Yamashita T. Ishiguro N. Kadomatsu K. N-acetylglucosamine 6-O-sulfotransferase-1-deficient mice show better functional recovery after spinal cord injury.J Neurosci. 2010; 30: 5937-5947Crossref PubMed Scopus (57) Google Scholar Expression of the 5D4 epitope was also found physiologically in the developing brain of rat and mouse.12Hoshino H. Foyez T. Ohtake-Niimi S. Takeda-Uchimura Y. Michikawa M. Kadomatsu K. Uchimura K. KSGal6ST is essential for the 6-sulfation of galactose within keratan sulfate in early postnatal brain.J Histochem Cytochem. 2014; 62: 145-156Crossref PubMed Scopus (22) Google Scholar, 22Meyer-Puttlitz B. Milev P. Junker E. Zimmer I. Margolis R.U. Margolis R.K. Chondroitin sulfate and chondroitin/keratan sulfate proteoglycans of nervous tissue: developmental changes of neurocan and phosphacan.J Neurochem. 1995; 65: 2327-2337Crossref PubMed Scopus (113) Google Scholar, 23Miller B. Sheppard A.M. Pearlman A.L. Developmental expression of keratan sulfate-like immunoreactivity distinguishes thalamic nuclei and cortical domains.J Comp Neurol. 1997; 380: 533-552Crossref PubMed Scopus (25) Google Scholar Expression of the 5D4 epitope in microglia is extensively induced with neurodegeneration in mouse models of cerebral amyloid angiopathy,24Miao J. Vitek M.P. Xu F. Previti M.L. Davis J. Van Nostrand W.E. Reducing cerebral microvascular amyloid-beta protein deposition diminishes regional neuroinflammation in vasculotropic mutant amyloid precursor protein transgenic mice.J Neurosci. 2005; 25: 6271-6277Crossref PubMed Scopus (53) Google Scholar, 25Fan R. DeFilippis K. Van Nostrand W.E. Induction of complement proteins in a mouse model for cerebral microvascular A beta deposition.J Neuroinflammation. 2007; 4: 22Crossref PubMed Scopus (34) Google Scholar familial Danish dementia,26Vidal R. Barbeito A.G. Miravalle L. Ghetti B. Cerebral amyloid angiopathy and parenchymal amyloid deposition in transgenic mice expressing the Danish mutant form of human BRI2.Brain Pathol. 2009; 19: 58-68Crossref PubMed Scopus (40) Google Scholar and light-induced retinal degeneration.27Zhang C. Shen J.K. Lam T.T. Zeng H.Y. Chiang S.K. Yang F. Tso M.O. Activation of microglia and chemokines in light-induced retinal degeneration.Mol Vis. 2005; 11: 887-895PubMed Google Scholar However, the functional role of 5D4-reactive KS in the pathogenesis of neurodegenerative disease is unknown. Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease that attacks spinal and bulbar motor neurons (lower motor neurons) in the spinal cord and brainstem and corticospinal and corticobulbar motor neurons (upper motor neurons) in the cerebral cortex, leading to progressive muscle weakness and complete paralysis within 1 to 5 years after disease onset.28Beghi E. Logroscino G. Chio A. Hardiman O. Mitchell D. Swingler R. Traynor B.J. EURLAS ConsortiumThe epidemiology of ALS and the role of population-based registries.Biochim Biophys Acta. 2006; 1762: 1150-1157Crossref PubMed Scopus (146) Google Scholar The mutation of expanded GGGGCC hexanucleotide repeat in the C9ORF72 gene is the most common cause of ALS. The repeat expansions are related to up to 40% of familial ALS and 10% of sporadic ALS in the United States and Europe.29Majounie E. Renton A.E. Mok K. Dopper E.G. Waite A. Rollinson S. et al.Frequency of the C9orf72 hexanucleotide repeat expansion in patients with amyotrophic lateral sclerosis and frontotemporal dementia: a cross-sectional study.Lancet Neurol. 2012; 11: 323-330Abstract Full Text Full Text PDF PubMed Scopus (753) Google Scholar Mutations in the gene encoding copper–zinc superoxide dismutase (SOD1) are found in 10% to 20% of familial ALS and up to 5% of sporadic ALS globally.30Rosen D.R. Mutations in Cu/Zn superoxide dismutase gene are associated with familial amyotrophic lateral sclerosis.Nature. 1993; 364: 362Crossref PubMed Scopus (24) Google Scholar, 31Boillee S. Vande Velde C. Cleveland D.W. ALS: a disease of motor neurons and their nonneuronal neighbors.Neuron. 2006; 52: 39-59Abstract Full Text Full Text PDF PubMed Scopus (1026) Google Scholar The SOD1G93A transgenic mouse is an ALS model mouse.32Gurney M.E. Pu H. Chiu A.Y. Dal Canto M.C. Polchow C.Y. Alexander D.D. et al.Motor neuron degeneration in mice that express a human Cu,Zn superoxide dismutase mutation.Science. 1994; 264: 1772-1775Crossref PubMed Scopus (3266) Google Scholar This mouse develops an ALS-like phenotype that includes loss of motor neurons in the spinal cord by the disease end stage.32Gurney M.E. Pu H. Chiu A.Y. Dal Canto M.C. Polchow C.Y. Alexander D.D. et al.Motor neuron degeneration in mice that express a human Cu,Zn superoxide dismutase mutation.Science. 1994; 264: 1772-1775Crossref PubMed Scopus (3266) Google Scholar It was reported that the lower and upper motor neurons degenerate independently of each other and that these degenerative processes are associated with intense inflammatory reactions in the spinal cord and brain.33Bruijn L.I. Miller T.M. Cleveland D.W. Unraveling the mechanisms involved in motor neuron degeneration in ALS.Annu Rev Neurosci. 2004; 27: 723-749Crossref PubMed Scopus (1140) Google Scholar New methods to monitor the progression of degenerative changes in the lower and upper motor neurons will help our understanding of causative mechanisms underlying ALS and aid to develop therapeutic strategies. Recently, we have shown that the 5D4 epitope is induced in microglia in the spinal cord of SOD1G93A mice with disease progression and that GlcNAc6ST1 is involved in 5D4-reactive KS synthesis.34Hirano K. Ohgomori T. Kobayashi K. Tanaka F. Matsumoto T. Natori T. Matsuyama Y. Uchimura K. Sakamoto K. Takeuchi H. Hirakawa A. Suzumura A. Sobue G. Ishiguro N. Imagama S. Kadomatsu K. Ablation of keratan sulfate accelerates early phase pathogenesis of ALS.PLoS One. 2013; 8: e66969Crossref PubMed Scopus (31) Google Scholar It was unclear whether the microglial 5D4-KS glycan is elicited in the brainstem and cerebral cortex of SOD1G93A mice. The sulfotransferase responsible for Gal-6-sulfation within the 5D4-KS in microglia of SOD1G93A mouse has remained unknown. Here, we report that the microglial 5D4-KS consists of sialyl-, fucosyl-, O-linked glycans, and that the 5D4-KS is expressed in the brainstem but not in the cerebral cortex of SOD1G93A mice. We have also found that KSGal6ST is an enzyme required for Gal-6-sulfation within the microglial 5D4-reactive KS in the spinal cord and brainstem of SOD1G93A mice, with a corroborating correlation between the expression level of GlcNAc6ST1 and the 5D4 epitope. C57BL/6J mice were purchased from SLC Inc. (Hamamatsu, Japan). ALS model B6.Cg-Tg(SOD1∗G93A)1Gur/J (SOD1G93A) mice32Gurney M.E. Pu H. Chiu A.Y. Dal Canto M.C. Polchow C.Y. Alexander D.D. et al.Motor neuron degeneration in mice that express a human Cu,Zn superoxide dismutase mutation.Science. 1994; 264: 1772-1775Crossref PubMed Scopus (3266) Google Scholar obtained from The Jackson Laboratory (Bar Harbor, ME) were provided by Dr. Akio Suzumura, Nagoya University, Japan. The generation of GlcNAc6ST1-deficient (GlcNAc6ST1−/−) or KSGal6ST-deficient (KSGal6ST−/−) mice was as previously described.35Uchimura K. Kadomatsu K. El-Fasakhany F.M. Singer M.S. Izawa M. Kannagi R. Takeda N. Rosen S.D. Muramatsu T. N-acetylglucosamine 6-O-sulfotransferase-1 regulates expression of L-selectin ligands and lymphocyte homing.J Biol Chem. 2004; 279: 35001-35008Crossref PubMed Scopus (71) Google Scholar, 36Patnode M.L. Yu S.Y. Cheng C.W. Ho M.Y. Tegesjo L. Sakuma K. Uchimura K. Khoo K.H. Kannagi R. Rosen S.D. KSGal6ST generates galactose-6-O-sulfate in high endothelial venules but does not contribute to L-selectin-dependent lymphocyte homing.Glycobiology. 2013; 23: 381-394Crossref PubMed Scopus (22) Google Scholar Males of SOD1G93A hemizygotes were bred with GlcNAc6ST1−/− or KSGal6ST−/− females. Generated SOD1G93A/GlcNAc6ST1+/− or SOD1G93A/KSGal6ST+/− males were then bred with GlcNAc6ST1+/− or KSGal6ST+/− females. The mice were kept under controlled environmental conditions and were provided with standard nourishment and water. All experimental procedures were approved by the Animal Research Committee of Nagoya University and conducted in accordance with the guidelines of Nagoya University. Colony maintenance and experimental tests for the SOD1G93A transgenic mice and the transgenic progeny were performed in accordance with guidelines provided by The Jackson Laboratory. The SOD1G93A transgene was genotyped with the genomic DNA isolated from a 0.5-cm section of the tail. Isolation of tail DNA was performed by a sodium hydroxide extraction method. The tail section cut into small pieces was mixed with 180 μL of 100 mmol/L NaOH. The sample was incubated at 95°C for 10 minutes, cooled to room temperature, and then neutralized with 20 μL of 1 mol/L Tris-HCl (pH 8.0). The sample was centrifuged at 15,000 × g for 10 minutes at 4°C. The supernatant fluid was subjected to PCR reactions. A total of 25 μL of the PCR mixture contained 0.25 μL of the tail DNA, 12.5 μL of 2× PCR buffer for KOD FX, 0.4 mmol/L of each dNTP, 5 μmol/L of each primer, 2.5% (v/v) dimethyl sulfoxide, and 0.5 units of KOD FX DNA polymerase (Toyobo, Osaka, Japan). The thermal cycling was set as follows: 94°C for 1.5 minutes, followed by 35 cycles of 94°C for 30 seconds, 60°C for 30 seconds, and 72°C for 30 seconds, and then 4°C until the samples were subjected to 1.5% agarose gel electrophoresis. The primers for the transgene (mutated SOD1) were 5′-CATCAGCCCTAATCCATCTGA-3′ (forward) and 5′-CGCGACTAACAATCAAAGTGA-3′ (reverse).37Alexander G.M. Erwin K.L. Byers N. Deitch J.S. Augelli B.J. Blankenhorn E.P. Heiman-Patterson T.D. Effect of transgene copy number on survival in the G93A SOD1 transgenic mouse model of ALS.Brain Res Mol Brain Res. 2004; 130: 7-15Crossref PubMed Scopus (121) Google Scholar Copy numbers of the transgene were determined with the use of real-time quantitative PCR (qPCR) in accordance with The Jackson Laboratory protocol run on Light Cycler 480 (Roche Diagnostics, Mannheim, Germany). The transgene copy number was determined by comparing the ΔCt value of each sample against a standard high-copy control and a low-copy control, with the use of appropriate endogenous references. All primers described in The Jackson Laboratory protocol were obtained from MBL (Nagoya, Japan). High-copy number transgenic mice were subjected to experimental analyses. Low-copy number transgenic mice were euthanized. The following materials were obtained from the commercial sources indicated. 5D4 anti-KS monoclonal antibody was from Seikagaku (Tokyo, Japan); rabbit anti–GlcNAc6ST-1/CHST2 antibody was from Sigma-Aldrich (St. Louis, MO); mouse anti-KSGal6ST/CHST1 antibody (clone A-2) was from Santa Cruz Biotechnologies (Dallas, TX); biotinylated rat anti-mouse Mac-2 was from Cedarlane (Burlington, NC); rabbit anti-Iba I antibody was from Wako (Osaka, Japan); horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG1 was from Caltag (Burlingame, CA); HRP-conjugated goat anti-mouse IgG2b, cyanine 2 (Cy2)-conjugated streptavidin, Cy3-conjugated goat anti-mouse IgG1, and Alexa Fluor 488-conjugated goat anti-rabbit IgG (H+L) were from Jackson ImmunoResearch (West Grove, PA); and HRP-conjugated goat anti-rabbit IgG (H+L) was from Cell Signaling Technology (Danvers, MA). Enzymes used for digestion were α2-3,6,8 neuraminidase (Arthrobacter ureafaciens) from Nacalai Tesque (Kyoto, Japan); α1-3,4-fucosidase (Streptomyces sp.) from Takara Bio Inc. (Shiga, Japan); keratanase II (Bacillus sp.) from Seikagaku; peptide-N-glycosidase F (PNGase F; Flavobacterium meningosepticum) from Roche Diagnostics (Indianapolis, Indiana); and α1-2 fucosidase (Xanthomonas manihotis), recombinant α-N-acetylgalactosaminidase, and α1-3,6 galactosidase (Xanthomonas manihotis) from New England Biolabs (Ipswich, MA). Patients with sporadic ALS received a pathologic diagnosis according to the El Escorial Diagnostic World Federation of Neurology criteria (Table 1). The diagnosis of ALS was histopathologically confirmed by the presence of intraneuronal Bunina bodies. Specimens of cervical (C3 to C6 segments) and lumbar spinal cords (L4 to L5 segments) from ALS cases and disease control cases were obtained. These specimens were frozen and then stored at −80°C. The collection of tissues, their use in this study, and the consent procedure were approved by the Ethics Committee of Nagoya University Graduate School of Medicine. The written informed consent was obtained from the patients' next-of-kin. Among the specimens examined, those with preserved expression of β-actin were subjected to evaluation of KS expression. Frozen spinal cords were homogenized with a Dounce tissue grinder in Tris-buffered saline (TBS; 20 mmol/L Tris and 137 mmol/L NaCl, pH 7.6) containing 1% Triton X-100 and protease inhibitors (complete protease inhibitor cocktail; Roche Diagnostics) and then used for biochemical analyses.Table 1Clinical Information of ALS Disease and Non-ALS Control Donor Patients Used in the Western Blot Analysis of the 5D4 Keratan Sulfate EpitopePatient numberAge, yearSexDiseaseALS disease duration, yearALS disease patients ALS-23061FALS5 ALS-23166MALS2.7 ALS-23353FALS2.7 ALS-26066MALS3Non-ALS controls Ctrl-13874FMSA-P Ctrl17777MPSP Ctrl-21684MMGALS, amyotrophic lateral sclerosis; F, female; M, male; MG, myasthenia gravis; MSA-P, multiple system atrophy with predominant parkinsonism; PSP, progressive supranuclear palsy. Open table in a new tab ALS, amyotrophic lateral sclerosis; F, female; M, male; MG, myasthenia gravis; MSA-P, multiple system atrophy with predominant parkinsonism; PSP, progressive supranuclear palsy. Mice were anesthetized and transcardially perfused with phosphate-buffered saline (PBS). Spinal cords and brains were dissected out and divided into cross-sectional segments and sagittal parts, respectively. Hemi-brains and segments of cervical, thoracic, and lumbar spinal cords were embedded in a compound for frozen sectioning. Thoracic and lumbar parts of spinal cords and regional parts of hemi-brains, namely, brainstem, thalamus, hippocampus, cerebellum, olfactory bulb, and the frontal motor region of cerebral cortex, were separated on ice, snap-frozen, and then stored at −80°C for biochemical analysis. Microglia were isolated from whole spinal cords of adult SOD1G93A and wild-type mice as previously described38Hickman S.E. Allison E.K. El Khoury J. Microglial dysfunction and defective beta-amyloid clearance pathways in aging Alzheimer's disease mice.J Neurosci. 2008; 28: 8354-8360Crossref PubMed Scopus (787) Google Scholar with minor modifications. SOD1G93A littermate mice with end-stage disease were deeply anesthetized and perfused transcardially with ice-cold PBS. Spinal cords were dissected out, cut into fragments with a scalpel, and then incubated with an enzyme solution that contained 5 mg/mL collagenase and 10 μg/μL DNase I (Roche Diagnostics) for 45 minutes at 37°C. The tissue was gently disrupted by pipetting several times with a 1-mL pipette, filtered through a 70-μm cell strainer, and then centrifuged at 190 × g for 10 minutes. The cell pellet was suspended with myelin magnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany) to remove myelin debris. The cells were then pelleted and re-suspended with PBS supplemented with 0.5% bovine serum albumin (BSA), 2 mmol/L EDTA, and CD11b magnetic beads (Miltenyi Biotec) for 15 minutes at 4°C. The suspension was subjected to magnetic-activated cell sorting selection columns according to the manufacturer's instructions. CD11b+ cells were collected and suspended in Dulbecco's modified Eagle's/F12 medium supplemented with 10% heat-inactivated fetal bovine serum. The cell suspension was seeded on a 6-well plate coated with poly-l-lysine (Sigma-Aldrich) and maintained in culture at 37°C. The medium was replaced every 2 days. Collected CD11b+ cells were characterized by qPCR, immunocytochemistry, and Western blot analysis. CD11b− cells in flow-through fractions on the mass spectrometry column were also collected and analyzed. Snap-frozen spinal cords and various regions of brains (approximately 20 mg) were taken in a 1.5-mL tube and supplemented with 600 μL (30 volumes of the tissue weight) of ice-cold TBS with protease inhibitors as previously described.39Hosono-Fukao T. Ohtake-Niimi S. Hoshino H. Britschgi M. Akatsu H. Hossain M.M. Nishitsuji K. van Kuppevelt T.H. Kimata K. Michikawa M. Wyss-Coray T. Uchimura K. Heparan sulfate subdomains that are degraded by Sulf accumulate in cerebral amyloid β plaques of Alzheimer's disease: evidence from mouse models and patients.Am J Pathol. 2012; 180: 2056-2067Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar The tube was then placed in the water bath of a Bioruptor sonicator (Cosmo Bio, Tokyo, Japan). Tissues were fragmented for 15 seconds with the maximum ultrasonic wave output four to five times until solid materials in the tube became invisible. The materials were then ultra-centrifuged at 100,000 × g for 30 minutes at 4°C. The supernatant fluid was collected and stored frozen as the TBS-soluble fraction. The resulting pellet was suspended in 600 μL (the same volume) of TBS that contained 1% SDS, and the pellet was dissociated and centrifuged at 15,000 × g for 20 minutes at room temperature. The resulting supernatant fluid was collected and stored frozen as the TBS-insoluble/1% SDS-soluble fraction. The protein concentrations of both fractions were measured by the Bradford method. Snap-frozen spinal cords were homogenized in TBS that contained 1% Triton X-100 and protease inhibitors. Supernatant fluids were collected after centrifugation at 10,000 × g for 30 minutes at 4°C. The protein concentration was measured by the Bradford method. For pretreatment, the samples were digested with 0.05 U/mL neuraminidase and/or 1 U/mL α-1,3/4-l-fucosidase at 37°C for 2 hours. Then, the enzymatic reaction was stopped by heating the samples at 95°C for 5 minutes. The samples were then treated with 0.05 U/mL keratanase II at 37°C overnight. Digestion of the samples with 1 U/mL PNGase F or β-elimination (GlycoProfile β-elimination kit; Sigma-Aldrich), α1-2 fucosidase, recombinant α-N-acetylgalactosaminidase, or α1-3,6 galactosidase was performed according to t

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